One of the critical events during the replication of many DNA viruses is the binding of "initiator proteins" to origins of replication. Once bound to the origins of replication, the virally encoded initiator proteins frequently assemble into higher oligomeric complexes. Our long-term goal is to develop a systematic approach for the isolation of inhibitors that will selectively block the assembly of viral initiators on origins of replication. These inhibitors will serve as useful reagents for exploring biological activity and as lead compounds for drug design. The systematic approach that we will utilize is an extension of recently described chemical genetics methods. A critical component of this technique is the use of split intein based vectors to generate diverse cyclic-peptide libraries in E. coli cells. This is a very cost effective method for generating complex, but very stable, libraries. Using straightforward genetic screens, the libraries will be searched for members that are able to disrupt the assembly of a prototype viral initiator termed Simian Virus 40 T-antigen. The advantages of selecting cyclic peptide inhibitors that block T-antigen oligomerization include the fact that much of T-ag's structure has been determined and T-ag's interaction with the Simian Virus 40 origin is understood in great detail. Furthermore, inhibitors of T-ag oligomerization are clinically relevant. For example, SV40 T-ag may be a human health issue; although interpretations remain uncertain, data have linked it to a variety of human cancers including mesothelioma and non-Hodgkin's lymphomas (reviewed in Vilchez and Butel (2004) Clinical Microbiology Reviews 17: 495-508). Furthermore, SV40 virus is closely related to two human viruses, BK and JC virus. These viruses induce a number of diseases in humans, including cancer. In addition, JC virus induces progressive multifocal leukoencephalopathy (PML); a disease that occurs in patients whose cellular immunity has been impaired. Indeed, approximately 5% of patients with AIDS have PML. Therefore, the approach that we propose to use to isolate inhibitors of T-ag assembly may identify lead compounds against BK and JC viruses. More importantly, these experiments will serve to demonstrate that the chemical genetic approach that we describe is a general method for the isolation of inhibitors against viral initiators. Once the feasibility of the approach is demonstrated, similar experiments will be conducted with the viral initiators encoded by other DNA viruses, such as those encoded by Herpes simplex virus and different strains of human papillomavirus. Thus, the approach described herein might facilitate the isolation of compounds with broad clinical relevance. 1 DNA viruses are a significant health risk; however, there are currently few options for treating these pathogens. Describe herein is a proposal for a chemical genetics approach for the isolation of inhibitors against DNA tumor viruses. Thus the proposal is likely to have broad clinical significance. [unreadable] [unreadable] [unreadable]